1. Field of the Invention
This invention relates to the analysis of various compounds in biological fluids or the like and, more particularly, to certain novel labeled analytes and to methods of analysis using such labeled analytes.
2. Description of the Prior Art
For a variety of clinical purposes such as, for example, monitoring dosage schedules, monitoring hormone levels, checking for recent ingestion or following pharmacological dynamics of bioavailability, absorption, degradation or excretion, it is a great advantage to measure the concentration of various drugs or the like to the nanomolar or even picomolar level. As is known, radioimmunoassay can accomplish analyses of this type. To carry out an analysis, an acceptable kit or system must include an antiserum, a standard or known concentration of the compound (i.e.--analyte) to be measured, a radio-labeled derivative of the compound to be measured and a buffering agent or agents. The antiserum is produced by bleeding animals which have been immunized by innoculation, for example, with the hapten - protein conjugate (immunogen) corresponding to the compound to be measured.
As is well known, the technique of radioimmunoassay, in general, measures the competition between radioactively labeled analyte and unlabeled analyte for binding sites on the antibody in the antiserum. By adding to the antiserum known amounts of the analytes to be assayed and a radiolabeled analog, a dose - response curve for bound or free analyte vs. concentration of analyte is constructed. After this immuno-calibration has been carried out, unknown concentrations can then be compared to the standard dose-response curve for assay. Crucial to this type of assay is the existence of radioactive analytes which compete effectively with non-radioactive analytes. Accordingly, in order to obtain the maximum precision, accuracy, sensitivity, specificity and reproducibility of the assay, purified, well-characterized synthetic radioactive analytes are required.
Several deficiencies in radioimmunoassay methodology have been identified. First of all, it is necessary to make a physicaI separation of the antibody-bound, radiolabeled analyte from the free radiolabeled analyte. Further, the methodology is considered rather labor intensive; and the equipment required is likewise relatively expensive, is not uniformly available and further requires the use of highly trained and skilled technicians to accurately carry out such assays. Likewise, the radioisotopically-labeled analytes are relatively unstable and expensive and pose an increasingly severe waste disposal problem owing to radiation exposure hazards associated with the commonly used radioisotopic labels. Despite these shortcomings, the use of radioimmunoassay has grown considerably.
The substantial recent growth in the use of radio-immunoassay in clinical laboratories has, however, spurred the development of variants which overcome the deficiencies of the radioimmunoassay methodology as described herein.. The approaches which have been developed to overcome these deficiencies primarily involve the use of enzyme or fluorescent labels instead of radioisotopic labels, preferably coupled with conditions allowing for measuring a chemical distinction between bound and free fractions of labeled analyte which leads to the elimination of the requirement for physical separation. Immunoassays having the latter simplifying and advantageous feature are referred to as homogeneous immunoassays as opposed to heterogeneous immunoassays where physical separation is required.
Thus, homogeneous immunoassay systems have been developed which are based on the use of an enzyme-labeled analyte where the enzymatic activity of the label is decreased when complexation with the antibody occurs. Unlabeled analyte whose concentration is to be determined displaces the enzymelabeled analyte bound to the antibody, thus causing an increase in enzymatic activity. Standard displacement or dose-response curves are constructed where increased enzymatic activity (monitored spectrophotometrically using what has been termed a "substrate" which ultimately produces a unique chromophore as a consequence of enzyme action) is plotted against increased analyte concentration. These are then used for determining unknown analyte concentrations. The following U.S. Pat. Nos. have been issued in the field of homogeneous enzyme immunoassay: 3,817,837; 3,852,157; 3,875,011; 3,966,556; 3,905,871; 4,065,354; 4,043,872; 4,040,907; 4,039,385; 4,046,636; 4,067,774; 4,191,613; and 4,171,244. In these patents, the label for the analyte is described as an enzyme having a molecular weight substantially greater than 5,000. Commercialization of this technology has been so far limited to applications where the analytes are relatively small in molecular size at fluid concentrations of the analyte greater than 10.sup.-10 M. These limitations result from the fact that the commonly used enzyme labels derived from large polypeptide analytes are not inhibited by binding to the anti-analyte antibody. Also, sensitivity limitations result from the lack of a fluorometric reporter molecule resulting from enzyme action and from the presence of serum interference at low concentrations, such as, for example, endogenous enzyme. Furthermore, the enzyme labels can be difficult to prepare reproducibly and to satisfactorily purify.
As a consequence of the limitations of the homogeneous enzyme immunoassay techniques described above, considerable effort has been devoted towards developing more sensitive homogeneous immunoassays using fluorescence. These have been primarily directed at assays for the larger sized molecules such as immunoglobulins or polypeptide hormones such as insulin. The following U.S. Pat. Nos. have been issued for this type of assay: 3,998,943; 3,996,345; 4,174,384; 4,161,515; 4,208,479 and 4,160,016. The label in most of these patents involves an aromatic fluorescent molecule bound either to the analyte or to the antibody. All likewise involve various methods of quenching fluorescence through antibodies or other fluorescent quenchers so that the extent of quenching is related to the amount of analyte present in the sample. Assays based on these approaches have not been commercialized probably owing to the difficulty in preparing satisfactorily purified fluorescent labeled-antibodies or analytes or related quencher-labeled species. Also, background fluorescence in serum may occur as well as serum-induced quenching. Still further, since such methods are not enzyme amplified, satisfactory sensitivity may be a problem.
Still other United States patents in this field which cannot be readily categorized in terms of the type of the immunoassay include: 3,935,074; 4,130,462; 4,160,645 and 4,193,983. The approach set forth in U.S. 4,160,645 includes the use of an electron transfer catalyst as a label. The catalyst (label) is deactivated by bonding to antibody.
Additional United States patents directed to the preparation of hapten conjugates to be used for the preparation of antibodies include: 3,884,898; 3,843,696; 4,045,420; 3,888,866; 3,917,582; 4,025,501; 4,043,989; 4,058,511; 4,069,105; 4,123,431; and 4,186,081. All of these patents relate to analyte derivatives and corresponding polypeptide conjugates wherein the polypeptide is antigenic and has a molecular weight in the range of 5,000 to 10.sup.6. Proteins such as albumin and globulin are specifically set forth.
Also, pretreatment methodologies for homogeneous enzyme immunoassays have been provided. U.S. Pat. Nos. in this area include: 3,856,469; 4,056,608 and 4,121,975.
A further type of methodology which may be described as a reactant-labeled fluorescent immunoassay involves the use of a fluorescent labeled analyte designed so that a fluorescent product is released when it is enzymatically hydrolzyed. Antibody to the analyte portion of the molecule, however, inhibits enzymatic hydrolysis. Consequently, by the law of mass action, fluorescence is enhanced in the presence of increased analyte due to enzymatic hydrolysis of the displaced, fluorescent labeled analyte. As an example, a labeled analyte is .beta.-galactosyl-umbelliferone-sisomicin. The enzyme .beta.-galactosidase cleaves the sugar from the umbelliferone moiety which can then fluoresce. Publications which describe this methodology include: J.F. Burd, R.C. Wong, J.E. Feeney, R.J. Carrico and R.C Boguolaski, Clin. Chem., 23, 1402(1977); Burd, Carrico, M.C. Fetter, et al., Anal. Biochem., 77, 56 (1977) and F. Kohen, Z. Hollander and Boguolaski, Jour. of Steroid Biochem., 11, 161 (1979).
Yet another type of homogeneous non-isotopic immunoassay is disclosed in U.S. Pat. No. 4,213,893, utilizing cofactor-labeled analytes. This involves labeling an analyte by linking it to a derivative of AND (i.e.--nicotinamide-6(2-aminoethylamino)-purine dinucleotide). The labeled cofactor retains its reactivity with dehydrogenases (e.g.--alcohol dehydrogenase, malate dehydrogenase, viz.--ADH, MDH) in cycling reactions for estriol as the analyte. Ultimately, the NADPH which is formed in these reactions is monitored fluorometrically and is a measure of the cycling rate. The NADPH is reduced in the presence of estriol antibody owing to complexation of the labeled cofactor. Thus, the cycling rate is directly related to the amount of estriol and was found to increase linearly with increasing amounts of estriol. This is described in a 1978 publication by F. Kohen, Z. Hollander, F. Yeager, R.J. Carrico, and R.C. Boguolaski, pp. 67-79, "Enzyme-labeled Immunoassay of Hormones and Drugs", edited by S.B. Pal, Walter de Gruiter, Berlin and New York. A similar system has been described for biotin and 2, 4-dinitrofluorobenzene analytes using lactic dehydrogenase and diaphorase as cycling enzymes (R.J. Carrico, J.E. Christner, R.C. Boguolaski and K.K. Young, Anal. Biochem., 72, 271 (1976)). It has been pointed out that the methodology may be subject to interference by endogenous co-factors and degrading enzymes common to bodily fluids (M.J. O'Sullivan, J.W. Bridges and V. Mark, Annals of Clinical Biochemistry, 19, 221 (1977)).
Yet another type of immunoassay technique utilizes an enzyme modulator as a label, viz.--an enzyme inhibitor or an allosteric effector. A number of enzyme modulators are listed along with their respective enzymes in U.S. Pat. No. 4,134,792. When a specific antibody binds to an enzyme modulator-labeled analyte, the enzyme modulator can no longer inhibit or otherwise affect the activity of an enzyme in the incubating mixture. Thus, displacement of the enzyme modulator-labeled analyte by free analyte restores inhibition or the allosteric effect of the enzyme modulator.
In a recent work entitled "Enzyme Immunoassay", published by Chemical Rubber Company, 1980, edited by Edward T. Maggio, the chapter entitled "Principles of Homogeneous Enzyme-Immunoassay" , pages 105-134, makes reference to the use of ribonuclease A as an enzyme label for human immunoglobulin G. Although details are not presented, the authors conclude that ribonuclease A has potential for use as a label in protein homogeneous enzyme-immunoassays. However, the authors also note that, unfortunately, the ubiquitous nature of ribonuclease A represents a serious potential for interference from endogenous enzyme in serum assays, and limits the practical utility of the procedure.
Further, considerable investigation of the structure and properties of ribonucleases has been carried out. For example, many organic compounds have been utilized heretofore for monitoring the catalytic activity of ribonuclease. Such organic compounds, or substrates, as they are ccmmonly referred to, include ribonucleic acid itself, cyclic phosphate diesters, and monoribonucleotide compounds which exhibit the same or similar structural constraints as those expressed by the natural substrate.
Still other compounds have been utilized for kinetically monitoring ribonuclease activities. Such compounds include 3'-uridylic acid phosphodiesters of 1-naphthol, 5-hydroxynaphthol, and 1-4 methoxyphenol, H. Rubsamen, R. Khandler, and H. Witzel (Hoppe-Seyler's Z. Physiol. Chem., 355, 687 (1974)). However, the hydrolysis product is monitored directly in the ultraviolet region, which is not sufficiently sensitive for analyses in the nanomolar or picomolar range and where interferences derived from clinical samples may occur. Further these substrates are difficult to prepare and require numerous steps, including lengthy chromatographic procedures.
Also, the cleavage of ribonucleases to polypeptide fragments has been investigated. For example, the action of subtilisin, a bacterial protease, on bovine pancreatic ribonuclease (ribonuclease A) is known. It has thus been found that a short 20 residue polypeptide and a long 104 residue polypeptide resulted as a consequence of the cleavage at the 20th peptide bond (counting from the amino terminus) of the ribonuclease A. The former is called the S-peptide while the latter is referred to as the S-protein (F.M. Richards, Proc. Nat'L Acad. Sci. U.S., 44 162 (1958); F.M. Richard and P.J. Vithayahil, J. Biol. Chem., 234, 1459 (1959)). It is likewise known that the two polypeptides readily combine to form a non-covalent complex (K=10.sup.-8 M) which retained the same catalytic activity of the original enzyme, ribonuclease A, towards a variety of substrates such as RNA or cytidine 2', 3'-phosphate diester. However, insofar as is known, this knowledge has not heretofore been utilized in developing immunoassay techniques.
Thus, despite the considerable recent activity in the field of homogeneous immunoassay, there remains the need for further development which can overcome various shortcomings of the presently used techniques. This is perhaps evident from the comparatively restricted commercial usage of non-isotopic immunoassay techniques despite the apparent broad potential. Thus, despite their well-recognized deficiencies, radioimmunoassay techniques continue to be widely used simply because satisfactory alternative techniques are not commercially available.
It is accordingly an object of the present invention to provide a homogeneous immunoassay technique which is broadly applicable to a wide range of analytes. A related and more specific object provides a homogeneous immunoassay technique which is thus not restricted to use with relatively small molecular weight analyte molecules.
A further object lies in the provision of a homogeneous immunoassay method which does not require the use of relatively high molecular weight labels. A related object is to provide labeled analytes which can be easily prepared, are readily purified and are relatively stable.
Another object of the present invention provides a homogeneous immunoassay method which is capable of operating in either a spectrophotometric or a fluorometric detection mode, using a substrate common to either mode, if desired.
A still further object of this invention is to provide a homogeneous immunoassay method which is capable of achieving superior sensitivity. A related and more specific object involves an assay with a fluorometric detection mode which is amplified by catalytic turnover of the substrate.
A still further object lies in the provision of a homogeneous immunoassay method which can be readily adapted for use in commercially available automatic analyzers, such as, for example, what are commonly termed "centrifugal fast analyzers".
Another object of the present invention is to provide a homogeneous immunoassay technique which is readily adaptable to automatic data reduction. A related and more specific object provides such an immunoassay technique wherein a dose-response curve satisfactory for automatic data reduction can be achieved.
A further object of this invention provides a homogeneous immunoassay technique wherein the methodology is sufficiently flexible to minimize problems such as, for example, potential interferences and the like.